Methods for inhibiting conversion of carnitine to trimethylamine (tma)

ABSTRACT

The invention provides a method of inhibiting the conversion of carnitine to trimethylamine (TMA) and lowering TMAO in an individual comprising administering to the individual one or more compositions comprising a compound set forth in Formula (I): 
     
       
         
         
             
             
         
       
     
     Wherein the compound is administered in an amount effective to inhibit conversion of carnitine to TMA in the individual.

FIELD OF THE INVENTION

The invention generally relates to materials and methods for inhibitingtrimethylamine production.

BACKGROUND

Trimethylamine (TMA) and its derivative trimethylamine-N-oxide (TMAO)are metabolites linked to disorders such as kidney disease, diabetesmellitus, trimethylaminuria, and cardiovascular disease (CVD). CVD is ageneral term encompassing a range of conditions affecting the heart andblood vessels, including atherosclerosis, coronary heart disease,cerebrovascular disease, heart failure, cardiomyopathy, atherothromboticdisease, aorto-iliac disease, and peripheral vascular disease. CVD isgenerally associated with conditions that involve narrowed, blocked,aneurysmal or dissection of one or more blood vessels, or thrombosis(blood clot formation). Complications associated with CVD include, butare not limited to, myocardial infarction, stroke, angina pectoris,acute coronary syndrome, transient ischemic attacks, congestive heartfailure, aortic aneurysm, atrial fibrillation or flutter, ventriculararrhythmias, cardiac conduction abnormalities, need forrevascularization and death. Revascularization can include but is notlimited to angioplasty, stenting, coronary artery bypass grafting,repair or replacement of vascular shunt or access such as anarteriovenous fistula. Complications associated with atherothromboticdisease include, but are not limited to, myocardial infarction, stroke,pulmonary embolism, deep venous thrombosis. According to the WorldHealth Organization, CVDs are the leading cause of death globally, withover 75% of deaths occurring in low- and middle-income countries. WorldHealth Organization Fact Sheet No. 317, updated January 2015. The WorldHealth Organization projects that diabetes will be the seventh leadingcause of death in 2030. World Health Organization Fact Sheet No. 312,updated January 2015. Prevention and management of conditions associatedwith TMA and TMAO, including CVD and diabetes, is a major public healthconcern.

SUMMARY OF THE INVENTION

The disclosure is based, at least in part, on the discovery thatcompounds of Formula (I), Formula (II), Formula (III), and Formula (IV),inhibit carnitine metabolism by gut microbiota, resulting in reductionin the formation of trimethylamine (TMA) and trimethylamine N-oxide(TMAO). The disclosure provides compositions and methods for, e.g.,inhibiting the conversion of carnitine to TMA in vitro and in vivo, forimproving or maintaining cardiovascular, cerebrovascular andperipherovascular health, and for improving or preventing a conditionassociated with TMA and TMAO.

In certain aspects, the invention provides one or more methods ofinhibiting the conversion of carnitine to trimethylamine (TMA) by abacterium comprising: contacting the bacterium with a compound as setforth in Formula (I):

wherein R₁ is selected from cyanate, isocyanate, thiocyanate,isothiocyanate, nitrile, isonitrile, or sulfhydryl; n′ is selected from0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and R₂ is selected from alkyl,branched alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, orsubstituted carbonyl; wherein when R₂ is phenyl, R₂ is substituted with0, 1, or 2 groups independently selected from alkyl, branched alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, halo, or aryl; with thecondition that when R₂ is heteroalkyl or heterocycloalkyl, theheteroatom(s) are not S; and the condition that when n′ is 2, R₂ is notunsubstituted phenyl.

In certain aspects, the invention provides one or more methods ofinhibiting the conversion of carnitine to trimethylamine (TMA) in anindividual. The method comprises administering to the individual one ormore compounds as set forth in Formula (I):

wherein R₁ is selected from cyanate, isocyanate, thiocyanate,isothiocyanate, nitrile, isonitrile, or sulfhydryl; n′ is selected from0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and R₂ is selected from alkyl,branched alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, orsubstituted carbonyl; wherein when R₂ is phenyl, R₂ is substituted with0, 1, or 2 groups independently selected from alkyl, branched alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, halo, or aryl; with thecondition that when R₂ is heteroalkyl or heterocycloalkyl, theheteroatom(s) are not S; and the condition that when n′ is 2, R₂ is notunsubstituted phenyl. The compound is administered in an amounteffective to inhibit conversion of carnitine to TMA and TMAO in theindividual.

In certain aspects, the invention provides one or more methods ofimproving a condition associated with the conversion of carnitine totrimethylamine by inhibiting the conversion of carnitine totrimethylamine (TMA) in an individual. The method comprisesadministering to the individual one or more compounds as set forth inFormula (I):

wherein R₁ is selected from cyanate, isocyanate, thiocyanate,isothiocyanate, nitrile, isonitrile, or sulfhydryl; n′ is selected from0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and R₂ is selected from alkyl,branched alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, orsubstituted carbonyl; wherein when R₂ is phenyl, R₂ is substituted with0, 1, or 2 groups independently selected from alkyl, branched alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, halo, or aryl; with thecondition that when R₂ is heteroalkyl or heterocycloalkyl, theheteroatom(s) are not S; and the condition that when n′ is 2, R₂ is notunsubstituted phenyl. The compound is administered in an amounteffective to treat or prevent the disease or condition associated withcarnitine-related trimethylamine (TMA) in the individual.

The invention further provides one or more methods of improving ormaintaining cardiovascular health. The method comprises administering tothe individual one or more compounds as set forth in Formula (I) anddescribed herein in an amount that improves or maintains cardiovascularhealth. The invention also provides one or more methods of improving acondition associated with the conversion of carnitine to trimethylamine(TMA) in an individual. The method comprises administering to theindividual one or more compositions comprising a compound as set forthin Formula (I) and described herein in an amount effective to improvethe condition. In some embodiments, the condition is trimethylaminuria,kidney disease, diabetes mellitus, or cardiovascular disease, e.g.,angina, arrhythmia, atherosclerosis, cardiomyopathy, congestive heartfailure, coronary artery disease (CAD), carotid artery disease,endocarditis, coronary thrombosis, myocardial infarction (MI), highblood pressure/hypertension, hypercholesterolemia/hyperlipidemia, atrialfibrillation or flutter, ventricular arrhythmias, cardiac conductionabnormalities, pulmonary embolism, deep venous thrombosis, peripheralartery disease (PAD), or stroke.

The invention further provides use of the compounds of Formula (I) forinhibiting the conversion of carnitine to TMA in vivo or in vitro, forimproving or maintaining cardiovascular health, and for improving acondition associated with the conversion of carnitine to TMA. Alsoprovided is the compound of Formula (I) for use in inhibiting theconversion of carnitine to TMA in vivo or in vitro, for improving ormaintaining cardiovascular health, and for improving a conditionassociated with the conversion of carnitine to TMA.

The foregoing summary is not intended to define every aspect of theinvention, and additional aspects are described in other sections, suchas the Detailed Description. In addition, the invention includes, as anadditional aspect, all embodiments of the invention narrower in scope inany way than the variations defined by specific paragraphs set forthherein. For example, certain aspects of the invention that are describedas a genus, and it should be understood that every member of a genus is,individually, an aspect of the invention. Also, aspects described as agenus or selecting a member of a genus should be understood to embracecombinations of two or more members of the genus. With respect toaspects of the invention described or claimed with “a” or “an,” itshould be understood that these terms mean “one or more” unless contextunambiguously requires a more restricted meaning. The term “or” shouldbe understood to encompass items in the alternative or together, unlesscontext unambiguously requires otherwise. If aspects of the inventionare described as “comprising” a feature, embodiments also arecontemplated “consisting of” or “consisting essentially of” the feature.

DETAILED DESCRIPTION OF THE INVENTION

Trimethylamine (TMA) synthesized by bacteria resident in the gut ofmammals is oxidized in the liver to trimethylamine N-oxide (TMAO).Exemplary precursors to TMA include carnitine, acylcarnitines,gamma-butyrobetaine, crotonobetaine, dehydrocarnitine, and TMAO, many ofwhich are derived from dietary sources such as, for example, dairyproducts and meats. Without wishing to be bound to a particularmechanism or biochemical pathway, the conversion of carnitine to TMA ismediated by an oxygenase/reductase, CntAB. Zhu et al., Proc. Natl. Acad.Sci. (2014), 111: 4268-4273. The reduction of carnitine conversion toTMA by bacteria in the gut of an individual leads to a reduction in TMAabsorption from the gut, leading to a subsequent reduction in plasmaTMAO following oxidation of TMA to TMAO by the Flavin Monooxygenaseenzymes (i.e. FMO3) in the liver. Wang et al., Nature (2011), 472:57-63. Lower plasma TMAO levels are related to a lower incidence ofmajor cardiovascular events in humans. Tang et al., NEJM (2013) 368:1575-1584. The conversion of carnitine to TMA in the gut of anindividual may occur via a multi-step process, for example, by atwo-step process via the metabolism of carnitine to gamma-butyrobetainefollowed by the metabolism of gamma butyrobetaine to TMA, facilitated byat least two functionally different bacteria. Koeth et al., CellMetabolism (2014), 20: 799-812. It will be appreciated that modulatingthe “conversion of carnitine to TMA” encompasses the conversion ofcarnitine-associated intermediates to TMA, including intermediates suchas, but not limited to, gamma-butyrobetaine, crotonobetaine,dehydrocarnitine (Koeth et al.; Kleber (1997) FEMS Microbiolo. Lett.147: 1-9), and TMAO.

All measurements referred to herein are made at about 22° C. to 25° C.(i.e. room temperature) unless otherwise specified.

As used herein the term “individual” includes both humans and othertypes of mammals sharing the TMAO pathway, such as domesticated animals,including but not limited to, domestic dogs (canines), cats (feline),horses, cows, ferrets, rabbits, pigs, rats, mice, gerbils, hamsters,horses, and the like.

A wide variety of individuals may wish to reduce the level of TMAproduced by bacteria in their digestive tract. For example, individualsdiagnosed with cardiovascular disease may be directed by a physician totake prescription drugs or effect lifestyle changes to modulate bloodcholesterol levels to reduce the risk of serious cardiovascular events.Other individuals not previously diagnosed with cardiovascular diseasebut who wish to improve or maintain cardiovascular health may also wishto reduce plasma TMAO levels by reducing the level of TMA produced bydigestive tract bacteria. As described further herein, a reduction inTMA (and, by extension, TMAO) is achieved by the compositions describedherein, which include, for example, a dietary supplement or drugcomprising isothiocyanates, such as the compounds of Formula (I),Formula (II), Formula (III), or Formula (IV).

As used herein, “dose” refers to a volume of medication, such as liquidmedication or oral dosage unit, containing an amount of a drug activesuitable for administration on a single occasion, according to soundmedical practice. A dose can be orally administered. In one example, adose can be a liquid medication and can be about 30 mL, in anotherexample about 25 mL, in another example about 20 mL, in another exampleabout 15 mL, and in another example about 10 mL. In another example, adose of liquid medication can be from about 10 mL to about 75 mL, inanother example from about 15 mL to about 50 mL, in another example fromabout 25 mL to about 40 mL, and in another example from about 28 mL toabout 35 mL. In another example, the dose can be a solid dosage form andcan be from about 5 g to about 25 mg, in another example from about 3 gto about 100 mg, in another example from about 2 g to about 250 mg, inanother example from about 1.6 g to about 500 mg, and in another examplefrom about 1 g to about 750 mg. In one example, the dose can be a soliddosage form wherein one dose is about 3 g and in another example onedose is about 1.6 g. The concentration of active ingredients can beadjusted to provide the proper doses of actives given the liquid dosesize. In one example, the dose is intended to be administered every 4hours, in another example every 6 hours, in another example every 8hours, and in another example every 12 hours.

As used herein, “medication” refers to medications, such aspharmaceuticals, including prescription medications, over-the-countermedications, behind-the-counter medications and combinations thereof. Insome examples, a medication can be a supplement which can containvitamins, minerals, and botanicals (VMS).

Medication compositions can be in any suitable form including liquidcompositions and solid oral dosage forms. Non limiting examples ofliquid compositions can include syrups including cough syrups,respiratory preparations including MSR cold/flu medication, beverages,supplemental water, foam compositions, gel compositions, particlessuspended in a liquid formulation, a solid in a gelatin or foam, salinewash and combinations thereof. Non-limiting examples of solid oraldosage forms can include tablets, capsules, caplets, sachets, sublingualdosage forms, buccal dosage forms, soft gels including Vicks® LiquiCaps™and other liquid filled capsules, dissolvable dosage forms includingdissolvable strips, films, gums including a center filled gum, gummiesincluding a center filled gummy, lozenges, edible foods, such as foodbars, center filled tablets, powder, granules, pellets, microspheres,nanospheres, beads, or nonpareils, and combinations thereof. Tablets caninclude compressed tablets, chewable tablets, dissolvable tablets, andthe like. Tablets can include compressed tablets, chewable tablets,dissolvable tablets, and the like. In some examples, the medication canbe applied to the skin, in an ointment such as Vicks® VapoRub®. In otherexamples, the medication can be inhaled, such as a nose spray orinhaler. In other examples, the medication can be in a drink, such as awarm beverage. In other examples, the medication can contain apharmaceutical active. In other examples, the medication does notcontain a pharmaceutical active and/or VMS but can alleviate symptomsand/or provide a health benefit at least in part, through the coolingsensation.

The medications can be in a form that is directly deliverable to themouth, throat, and/or skin. In some example, the medication compositionscan be delivered by a delivery device selected from droppers, pump,sprayers, liquid dropper, saline wash delivered via nasal passageway,cup, bottle, canister, pressurized sprayers, atomizers, air inhalationdevices, squeezable sachets, power shots, blister cards, and otherpackaging and equipment, and combinations thereof. The sprayer,atomizer, and air inhalation devices can be associated with a battery orelectric power source.

The disclosure provides, e.g., one or more methods of inhibiting theconversion of carnitine to trimethylamine (TMA), one or more methods ofimproving cardiovascular health, and one or more methods of improving acondition associated with conversion of carnitine to trimethylamine(TMA) comprising administering to the individual one or morecompositions comprising a compound of Formula (I), Formula (II), Formula(III), or Formula (IV). Features of the compositions and methods aredescribed below. Section headings are for convenience of reading and notintended to be limiting per se. The entire document is intended to berelated as a unified disclosure, and it should be understood that allcombinations of features described herein are contemplated, even if thecombination of features are not found together in the same sentence, orparagraph, or section of this document. It will be understood that anyfeature of the methods or compounds described herein can be deleted,combined with, or substituted for, in whole or part, any other featuredescribed herein.

Compounds

The method of the disclosure includes administering to the individualone or more compositions comprising a compound set forth in Formula (I):

wherein R₁ is selected from cyanate, isocyanate, thiocyanate,isothiocyanate, nitrile, isonitrile, or sulfhydryl; n′ is selected from0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and R₂ is selected from alkyl,branched alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, orsubstituted carbonyl; wherein when R₂ is phenyl, R₂ is substituted with0, 1, or 2 groups independently selected from alkyl, branched alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, halo, or aryl; with thecondition that when R₂ is heteroalkyl or heterocycloalkyl, theheteroatom(s) are not S; and the condition that when n′ is 2, R₂ is notunsubstituted phenyl. The compound is administered in an amounteffective to achieve the desired effect, e.g., inhibit conversion ofcarnitine to TMA, improve or maintain cardiovascular health, and/orimprove a condition associated with conversion of carnitine to TMA.

In some cases, R₂ is selected from methyl, ethyl, propyl (such asn-propyl or isopropyl), butyl (such as n-butyl, isobutyl, sec-butyl, ort-butyl), pentyl (e.g., 1-pentyl, 3-pentyl, 3-methylbutyl,2-methylbutyl), hexyl (e.g., 1-hexyl), heptyl, octyl, nonyl, decyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, norbornyl, morpholino, piperidino, alkylamino (e.g.,trialkylammonium, 3-(diethylamino)propyl, dimethylamino, diethylamino,(t-butoxycarbonyl)amino, ((t-butoxycarbonyl)amino)butyl), phenyl,substituted phenyl, naphthyl, arylcarbonyl (e.g., benzoyl),alkylcarbonyl, carboxy, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, benzhydryl, and alpha-methylbenzyl. In some cases,R₂ is phenyl substituted with 1 or 2 groups selected from methyl, ethyl,propyl, butyl, alkoxy (e.g., methoxy, ethoxy), alkylthio (e.g.,methylthio, ethylthio), fluoro, bromo, chloro, iodo,(t-butoxycarbonyl)amino, or

In various embodiments, R₁ is an isothiocyanate. In some cases, thecompound is selected from the group consisting of benzhydrylisothiocyanate, (S)-(+)-alpha-methylbenzyl isothiocyanate, and benzylisothiocyanate. In various aspects of the invention, when R₁ is anisothiocyanate, n′ is 0 (i.e., CH₂ is absent). In some cases, thecompound is selected from the group consisting of cyclohexylisothiocyanate, 4-bromophenyl isothiocyanate, 4-chlorophenylisothiocyanate, phenyl isothiocyanate, 3-methoxyphenyl isothiocyanate,4-methoxyphenyl isothiocyanate, 4-(methylthio) phenyl isothiocyanate,m-tolyl isothiocyanate, and 1-napthyl isothiocyanate.

In various embodiments when R₁ is an isothiocyanate, n′ is at least 1(e.g., n′ is at least 1, at least 2, at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, or at least 10). Anadditional example of a compound of Formula (I) is 2-(4-chlorophenethyl)isothiocyanate.

Formula (I) also includes one or more salts or solvates of any compoundencompassed by Formula (I).

In various embodiments, the method of the disclosure comprisesadministering to the individual one or more compositions comprising acompound of Formula (I) as set forth in Formula (II):

wherein R₂ is as defined for Formula (I). The compound is administeredin an amount effective to inhibit conversion of carnitine to TMA in theindividual. In some cases, R₂ is selected from phenyl and substitutedphenyl (e.g., phenyl substituted with 1 or 2 groups independentlyselected from methyl, ethyl, methoxy, methylthio, bromo, chloro, or(t-butoxycarbonyl)amino). In some cases the compound is selected from4-methoxyphenyl isothiocyanate, phenyl isothiocyanate, 3-methoxyphenylisothiocyanate, or 4-(methylthio)phenyl isothiocyanate.Formula (II) also includes one or more salts or solvates of any compoundencompassed by Formula (II).

In various embodiments, the methods of the disclosure compriseadministering to the individual one or more compositions comprising acompound of Formula (I) as set forth in Formula (III):

wherein n′ and R₂ are as defined for Formula (I). The compound isadministered in an amount effective to inhibit conversion of carnitineto TMA in the individual. In some cases, R₂ is haloaryl (e.g.,halophenyl) and n′ is 2. In some cases, the compound is2-(4-chlorophenethyl) isothiocyanate.Formula (III) also includes one or more salts or solvates of anycompound encompassed by Formula (III).

In various aspects, the methods of the disclosure comprise administeringto the individual one or more compositions comprising a compound ofFormula (I) as set forth in Formula (IV):

wherein R₃ is selected from hydrogen, alkyl, or aryl; and R₄ is aryl;wherein when R₄ is phenyl, R₄ is substituted with 0, 1, or 2 groupsindependently selected from alkyl, branched alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, halo, or aryl; with the condition thatwhen R₄ is substituted with heteroalkyl or heterocycloalkyl, theheteroatom(s) are not S. The compound is administered in an amounteffective to inhibit conversion of carnitine to TMA in the individual.In some cases, R₃ is selected from methyl, ethyl, propyl, butyl, pentyl,phenyl, or substituted phenyl, such as phenyl substituted with 1 or 2groups selected from methyl, ethyl, propyl, butyl, alkoxy (e.g.,methoxy, ethoxy), alkylthio (e.g., methylthio, ethylthio), fluoro,bromo, chloro, iodo, or (t-butoxycarbonyl)amino). In some cases, R₄ isselected from phenyl or substituted phenyl, such as phenyl substitutedwith 1 or 2 groups selected from methyl, ethyl, propyl, butyl, alkoxy(e.g., methoxy, ethoxy), alkylthio (e.g., methylthio, ethylthio),fluoro, bromo, chloro, iodo, or (t-butoxycarbonyl)amino.Formula (IV) also includes one or more salts or solvates of any compoundencompassed by Formula (IV).

“Alkyl” refers to straight chained and branched saturated hydrocarbongroups containing 1-30 carbon atoms (i.e., C₁-C₃₀), for example, 1-20carbon atoms (i.e., C₁-C₂₀) or 1-10 carbon atoms (i.e., C₁-C₁₀). Invarious embodiments, the alkyl groups of R₂ and R₃ are independentlyselected from C₁-C₇ alkyls, i.e., alkyl groups having a number of carbonatoms encompassing the entire range (i.e., 1 to 7 carbon atoms), as wellas all subgroups (e.g., 1-3, 1-6, 2-7, 1-5, 3-6, 5-7, 1, 2, 3, 4, 5, 6,and 7 carbon atoms). Nonlimiting examples of alkyl groups includemethyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl (2-methylpropyl),t-butyl (1,1-dimethylethyl), 3,3-dimethylpentyl, and 2-ethylhexyl.Unless otherwise indicated, an alkyl group can be an unsubstituted alkylgroup or a substituted alkyl group. Alkyl groups optionally can besubstituted, for example, with one or more of hydroxy (OH), thiol (SH),aryl, heteroaryl, cycloalkyl, heterocyclyl, and amino. R₂ and/or R₃ maycomprise a heteroalkyl so long as the heteroatom is not sulfur.

The term “heteroalkyl” is defined similarly as alkyl except the carbonchain contains one to three heteroatoms, such as heteroatomsindependently selected from oxygen, nitrogen, or sulfur. Non-limitingexamples of heteroalkyl include ethers, esters, ketones, primary amines,secondary amines, tertiary amines and quaternary amines, amides,sulfhydryls, alkyl sulfides, or carbamates. Unless otherwise indicated,a heteroalkyl group can be an unsubstituted heteroalkyl group or asubstituted heteroalkyl group.

The term “cycloalkyl” refers to an aliphatic cyclic hydrocarbon groupcontaining 3-8 carbon atoms (e.g., 3-5, 5-8, 3, 4, 5, 6, 7, or 8 carbonatoms). Nonlimiting examples of cycloalkyl groups include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Unlessotherwise indicated, a cycloalkyl group can be an unsubstitutedcycloalkyl group or a substituted cycloalkyl group.

The term “heterocycloalkyl” is defined similarly as cycloalkyl, exceptthe ring contains one to three heteroatoms independently selected fromoxygen, nitrogen, or sulfur. Nonlimiting examples of heterocycloalkylgroups include piperdine, tetrahydrofuran, tetrahydropyran, 4H-pyran,dihydrofuran, morpholine, thiophene, 1,4-dioxane, furan, pyrrole,pyrrolidine, imidazole, pyrazole, triazole, thiazole, pyrazine, pyran,oxazole, oxazine, thiazine, pyrimidine, piridazine, thiine, and thelike. Cycloalkyl and heterocycloalkyl groups can be saturated orpartially unsaturated ring systems optionally substituted with, forexample, one to three groups, independently selected alkyl, alkyleneOH,C(O)NH₂, NH₂, oxo (═O), aryl, haloalkyl, halo, and OH. Heterocycloalkylgroups optionally can be further N-substituted with alkyl, hydroxyalkyl,alkylenearyl, and alkyleneheteroaryl.

The term “hydroxy” or “hydroxyl” refers to a “—OH” group. The term“amino” or “amine” refers to a —NH₂, or a —NH— group, wherein eachhydrogen in each of Formula (I), Formula (II), Formula (III), or Formula(IV), can be replaced with an alkyl, cycloalkyl, aryl, heteroaryl, orheterocycloalkyl group. “Amine” includes cyclic amines optionallysubstituted with one or more additional heteroatoms. The term “carboxy”or “carboxyl” refers to a “—COOH” group. The term “thiol” or“sulfhydryl” refers to a “—SH” group. The term “cyano” refers to a —C≡Ngroup, also designated —CN. The term “isocyanyl” refers to a —N≡C group.The term “isocyano” refers to a —N═C═O group. The term “isothiocyano”refers to a —N═C═S group. The term “nitro” refers to a —NO₂ group.

A “substituted” alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl,heteroaryl, or alkoxyl refers to an alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, aryl, heteroaryl, or alkoxyl having at least one hydrogenradical that is substituted with a non-hydrogen radical (i.e., asubstituent). Examples of non-hydrogen radicals (or substituents)include, but are not limited to, alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, ether, aryl, heteroaryl, heterocycloalkyl,hydroxyl, oxy (or oxo), alkoxyl, ester, thioester, acyl, carboxyl,cyano, nitro, amino, amido, or sulfur. When a substituted alkyl groupincludes more than one non-hydrogen radical, the substituents can bebound to the same carbon or two or more different carbon atoms.

Salts or solvates, e.g., physiologically acceptable salts, of thedisclosed compounds are contemplated and optionally are prepared byreacting the appropriate base or acid with a stoichiometric equivalentof the compound. Acids commonly employed to form physiologicallyacceptable salts include but are not limited to inorganic acids such ashydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid and phosphoric acid, as well as organic mono- di-and tri-acids such as para-toluenesulfonic acid, salicylic acid,tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylicacid, fumaric acid, gluconic acid, glucuronic acid, formic acid,glutamic acid, methanesulfonic acid, ethanesulfonic acid,benzenesulfonic acid, lactic acid, oxalic acid, malonic acid,para-bromophenylsulfonic acid, carbonic acid, succinic acid, glutaricacid, adipic acid, citric acid, benzoic acid and acetic acid, as well asrelated inorganic and organic acids. Physiologically acceptable saltsinclude sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, trifluoromethanesulfonate (ortriflate), acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate,terephthalate, sulfonate, xylene sulfonate, phenylacetate,phenylpropionate, phenylbutyrate, citrate, lactate, O-hydroxybutyrate,glycolate, maleate, tartrate, methanesulfonate, propanesulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and othersalts. Physiologically acceptable acid addition salts include, e.g.,those formed with mineral acids such as hydrochloric acid andhydrobromic acid and those formed with organic acids such as maleicacid.

Physiologically acceptable base addition salts may be formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Physiologically acceptable salts of compounds may also be prepared witha physiologically acceptable cation. Suitable physiologically acceptablecations are well known in the art and include but are not limited toalkaline, alkaline earth, ammonium and quaternary ammonium cations.Carbonates or hydrogen carbonates are also options in this regard.Examples of metals used as cations are sodium, potassium, magnesium,ammonium, calcium, ferric, and the like. Examples of suitable aminesinclude, but are not limited to, isopropylamine, histidine,N,N′-dibenzylethylenediamine, chloroprocaine, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine.

In some aspects of the invention, the compound is not naturally found incruciferous vegetables (e.g., broccoli).

In various embodiments, the compound of Formula (I), Formula (II),Formula (III), or Formula (IV) demonstrates an IC₅₀ of 1×10⁻³ or less,5×10⁻³ or less, 1×10⁻⁴ or less, 5×10⁻⁴ or less, 1×10⁻⁵ or less, 5×10⁻⁵or less, or 1×10⁻⁶ or less, or between 1×10⁻⁶ and 1×10⁻³, between 1×10⁻⁶and 1×10⁻⁴, between 1×10⁻⁶ and 1×10⁻⁵, between 1×10⁻⁵ and 1×10⁻³, orbetween 1×10⁻⁴ and 1×10⁻³ (observed 50% inhibition of TMA (or TMAO)formation from carnitine; mol/L), optionally in the assay described inthe Examples.

Methods

The invention includes one or more methods of inhibiting the conversionof carnitine to trimethylamine (TMA) in an individual comprisingadministering to the individual one or more compositions comprising acompound set forth in Formula (I), Formula (II), Formula (III), orFormula (IV), as described above under the subheading “Compounds.” Theindividual of any of the embodiments described herein is a mammal,preferably a human, such as a human in need of reduced TMA levels,improvement of cardiovascular health, and the like. Optionally, theindividual exhibits an elevated level of TMA or a metabolite thereof(e.g., TMAO, dimethylamine (DMA), or methylamine (MA, also known asmonomethylamine or MMA)) prior to administration. In variousembodiments, the individual suffers from cardiovascular disease, ingestsa diet high in carnitine, or exhibits one or more CVD risk factors(e.g., smoking, stress, high total cholesterol, high LDL cholesterol,low HDL cholesterol, age, hypertension, family history of CVD, obesity,prediabetes, and/or diabetes).

One or more methods of inhibiting the conversion of carnitine to TMA invitro also is contemplated. In this regard, the method comprisescontacting a bacterium (e.g., a bacterium that is represented in the gutmicrobiota) or a bacterial lysate that metabolizes carnitine to produceTMA with a compound of Formula (I), Formula (II), Formula (III), orFormula (IV), as described above under the subheading “Compounds.” Invarious embodiments, the bacterium is selected from Proteus mirabilis,Proteus penneri, Clostridium ljungdahlii, C. scindens, C. aldenense, C.aminobutyricum, Collinsella tanakaei, Anaerococcus vaginalis,Eggerthella lenta, Edwardsiella tarda, Streptococcus dysgalactiae,Desultitobacterium hafniense, Klebsiella variicola, K. pneumonia,Escherichia coli, E. fergusonii, or a combination thereof. Thedisclosure further provides one or more methods of identifying acompound that inhibits TMA production. A method comprises contacting abacterium (e.g., a bacterium that is part of the gut microbiota) or abacterial lysate that metabolizes carnitine to produce TMA with acandidate compound (e.g., a compound of Formula (I), Formula (II),Formula (III), or Formula (IV), as described above under the subheading“Compounds”), and detecting TMA (or a metabolite thereof). Optionally,the level of TMA (or metabolite thereof) produced by the bacterium incontact with the candidate compound is compared to (a) the level of TMAproduced by a bacterium or lysate not contacted with a candidatecompound or known TMA inhibitor or (b) the level of TMA produced by thebacterium prior to contact with the candidate compound. A reduction inthe level of TMA produced by the bacterium indicates that the candidatecompound inhibits conversion of carnitine to TMA.

One or more methods of inhibiting the conversion of carnitine to TMA invitro is also contemplated, wherein in certain embodiments a methodcomprises contacting bacteria or bacterial lysate with a compound ofFormula (I), Formula (II), Formula (III), or Formula (IV). In variousembodiments, the bacteria comprises a single bacterial species orstrain, or contains a mixture of two or more (e.g., three, four, five,or more) different bacterial species or bacterial strains. Similarly,the bacterial lysate is generated from a single bacteria species orstrain or multiple different bacterial species or strains.

It will be appreciated that “inhibiting conversion of carnitine to TMA”does not require complete elimination of TMA production via carnitinemetabolism. Any reduction in TMA formation from carnitine or a carnitinerelated metabolite as a precursor is contemplated, e.g., at least 1%, atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or 100% reduction;or from about 1% to about 100%, about 10% to about 90%, about 20% toabout 80%, about 30% to about 70%, about 40% to about 60%; or any othernumerical range which is narrower and which falls within such broadernumerical range, as if such narrower numerical ranges were all expresslywritten herein.

Any suitable method for measuring TMA in vitro or in vivo can be used inthe context of the invention. TMA, metabolites of TMA (e.g., TMAO, DMA,or MA), stable isotopes of TMA (e.g., deuterium labeled TMA, such asd3-, d6-, or d9-TMA), stable isotopes of TMAO (e.g., deuterium labeledTMAO, such as d3-, d6-, or d9-TMAO), stable isotopes of DMA (e.g.,deuterium labeled DMA, such as d3- or d6-DMA), stable isotopes of MA(e.g., deuterium labeled MA, such as d3-MA), and/or carnitine (includingstable isotopes of carnitine, for example d9-carnitine) can be assessedquantitatively or qualitatively. Exemplary methods of detecting andquantifying TMA are described in, e.g., U.S. Pub. No. 2010/00285517, thedisclosure of which is incorporated herein by reference in its entirety.For example, levels of TMA (or trimethylamine-N-oxide (TMAO), DMA, orMA) and/or carnitine are optionally measured via mass spectrometry,ultraviolet spectroscopy, or nuclear magnetic resonance spectroscopy.Mass spectrometers include an ionizing source (e.g., electrosprayionization), an analyzer to separate the ions formed in the ionizationsource according to their mass-to-charge (m/z) ratios, and a detectorfor the charged ions. In tandem mass spectrometry, two or more analyzersare included. Such methods are standard in the art and include, forexample, HPLC with on-line electrospray ionization (ESI) and tandem massspectrometry.

In various embodiments, TMA and/or TMAO is measured in a biologicalsample from an individual. Biological samples include, but are notlimited to, whole blood, plasma, serum, urine, feces, saliva, sweat,and/or tissue. The sample may be collected using anyclinically-acceptable practice and, if desired, diluted in anappropriate buffer solution, heparinized, concentrated, or fractionated.Any of a number of aqueous buffer solutions at physiological pH, such asphosphate, Tris, or the like, can be used. Acidified buffers also may beused. For example, the final pH after adding buffer to sample isoptionally between pH 1 and pH 6, e.g., between pH 1.5 and pH 3.0.

Optionally, levels of TMA (or a metabolite or stable isotope thereof)and/or carnitine in the biological sample is compared to a controlvalue. The control value utilized will depend on the embodiment of theinvention. In one aspect, the control value is the level of TMA and/orTMAO produced in the individual (or by the bacterium) prior toadministration or exposure to the compound of Formula (I), Formula (II),Formula (III), or Formula (IV). Alternatively, the control value isbased on levels measured in comparable samples obtained from a referencecohort (e.g., the general population, individuals diagnosed with a CVDor other TMA-associated condition, individuals not previously diagnosedwith a TMA-associated condition, nonsmokers, and the like). Levels ofTMA and/or TMAO and/or carnitine may be compared to a single controlvalue or to a range of control values. An individual is optionallyidentified as having an enhanced or elevated level of TMA prior toadministration by comparing the amount of TMA in a biological samplefrom the individual with a control value.

The invention further provides one or more methods of improvingcardiovascular health of an individual. In certain embodiments a methodcomprises administering to the individual one or more compositionscomprising a compound set forth in Formula (I), Formula (II), Formula(III), or Formula (IV), as described above under the subheading“Compounds” in an amount effective to improve cardiovascular health.Cardiovascular health is assessed by testing arterial elasticity, bloodpressure, ankle/brachial index, electrocardiogram, ventricularultrasound, platelet function (i.e. platelet aggregation), andblood/urine tests to measure, e.g., cholesterol, albumin excretion,C-reactive protein, or plasma B-type peptide (BNP) concentration. Invarious aspects of the invention, administration of the compound ofFormula (I), Formula (II), Formula (III), or Formula (IV), improves ormaintains one or more of the assay outcomes within normal ranges. Normalranges of outcomes of each test are known in the art. Improvement incardiovascular health is, in some embodiments, marked by a reduction incirculating total cholesterol levels, reduction in circulating lowdensity lipoproteins (LDLs), reduction in circulating triglycerides,and/or reduction in blood pressure.

The invention also includes one or more methods of improving a conditionassociated with conversion of carnitine to trimethylamine (TMA) in anindividual in need thereof. In certain embodiments a method comprisesadministering to the individual one or more compositions comprising acompound of Formula (I), Formula II, Formula (III), or Formula (IV), asdescribed above under the subheading “Compounds” in an amount effectiveto improve the condition. “Improving a condition” refers to anyreduction in the severity and/or onset of symptoms associated with adisorder caused, at least in part, by TMA. One of ordinary skill in theart will appreciate that any degree of protection from, or ameliorationof, a TMA-related disorder or symptom associated therewith is beneficialto an individual, such as a human. The quality of life of an individualis improved by reducing to any degree the severity of symptoms in anindividual and/or delaying the appearance of symptoms. Accordingly, themethod in one aspect is performed as soon as possible after it has beendetermined that an individual is at risk for developing a TMA-relateddisorder or as soon as possible after a TMA-related disorder isdetected.

The condition associated with the conversion of carnitine totrimethylamine is, in various aspects of the invention, a cardiovasculardisease, reduced or impaired kidney function, chronic kidney disease,end stage renal disease, trimethylaminuria, or diabetes mellitus. Theterm “cardiovascular disease” (CVD) is used in the art in reference toconditions affecting the heart, heart valves, and vasculature (e.g.,arteries and veins) of the body and encompasses diseases and conditionsincluding, but not limited to, arteriosclerosis, atherosclerosis,myocardial infarction, acute coronary syndrome, angina, congestive heartfailure, aortic aneurysm, aortic dissection, iliac or femoral aneurysm,pulmonary embolism, primary hypertension, atrial fibrillation, stroke,transient ischemic attack, systolic dysfunction, diastolic dysfunction,myocarditis, atrial tachycardia, ventricular fibrillation, endocarditis,arteriopathy, vasculitis, atherosclerotic plaque, vulnerable plaque,acute coronary syndrome, acute ischemic attack, sudden cardiac death,peripheral vascular disease, coronary artery disease (CAD), peripheralartery disease (PAD), and cerebrovascular disease.

In one aspect, the condition is atherosclerosis. Atherosclerosisinvolves the formation of atheromatous plaques that lead to narrowing(“stenosis’) of the vasculature, which can ultimately lead to partial orcomplete occlusion or rupture (aneurism) of the vessel, heart failure,aortic dissection, and ischemic events such as myocardial infarction andstroke. In various non-limiting embodiments, the inventive methodinhibits, reduces, or reverses (in whole or in part) the onset orprogression of atherosclerosis (e.g., reducing or preventing hardeningor thickening of the arteries, plaque formation, endothelium damage,and/or arterial inflammation).

In various embodiments, administration of the compound of Formula (I),Formula (II) or Formula (III), or Formula (IV), results in reduced TMAand/or TMAO levels, reduced total cholesterol levels, reduced LDLlevels, increased HDL levels, reduced triglyceride levels, and/ornormalized levels of other biomarkers associated with CVD (e.g.,excreted albumin, C-reactive protein, or plasma B-type peptide (BNP)).In some embodiments, the compound of Formula (I), Formula (II), Formula(III), or Formula (IV), reduces the risk of cardiovascular disease,reduced or impaired kidney function, chronic kidney disease, end stagerenal disease, trimethylaminuria, or diabetes mellitus when administeredto an individual.

Administration Regimens and Compositions

The amount of compound administered to the individual is sufficient toinhibit (in whole or in part) formation of TMA from carnitine. Invarious aspects of the disclosure, the amount improves cardiovascularhealth and/or achieves a beneficial biological response with respect toan unwanted condition associated with TMA (e.g., the amount issufficient to ameliorate, slow the progression, or prevent a condition(e.g., CVD)). The effect can be detected by, for example, an improvementin clinical condition, reduction in symptoms, or by any of the assays orclinical diagnostic tests described herein. The precise effective amountfor an individual can depend upon the individual's body weight, size,and health; the nature and extent of the condition; and the compound orcombination of agents selected for administration. In various aspects,the amount of compound administered to the individual is about 0.001mg/kg to about 1000 mg/kg. Specific ranges of doses in mg/kg includeabout 0.1 mg/kg to about 500 mg/kg, about 0.5 mg/kg to about 200 mg/kg,about 1 mg/kg to about 100 mg/kg, about 2 mg/kg to about 50 mg/kg, andabout 5 mg/kg to about 30 mg/kg. An effective amount may be administeredto the individual as a single deployment of compound or as a divideddoses (i.e., a single dose administered in multiple subunitscontemporaneously or close in time). An amount of compound is optionallydelivered one, two, or three times a day; one, two, or three times aweek; or one, two, three, or four times a month. The compound may bedelivered as a prodrug which is converted to an active drug in vitro orin vivo.

The compound or composition comprising the compound is administered byany route that allows inhibition of carnitine conversion to TMA. Thecompound or composition comprising the compound is, in various aspectsof the invention, delivered to an individual parenterally (e.g.,intravenously, intraperitoneally, intrapulmonary, subcutaneously orintramuscularly), intrathecally, topically, transdermally, rectally,orally, sublingually, nasally or by inhalation. In various preferredembodiments, the compound is administered to the gastrointestinal tractvia, e.g., ingestion. Sustained release formulations may also beemployed to achieve a controlled release of the compound when in contactwith body fluids in the gastrointestinal tract. Sustained releaseformulations are known in the art, and typically include a polymermatrix of a biological degradable polymer, a water-soluble polymer, or amixture of both, optionally with suitable surfactants.

The invention provides one or more compositions comprising the compoundof Formula (I), Formula (II), Formula (III), or Formula (IV), formulatedwith one or more physiologically acceptable excipients, carriers,stabilizers, or diluent for use in the methods described herein.Excipients include, but are not limited to, carrier molecules thatinclude large, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, antioxidants (e.g., ascorbic acid),chelating agents (e.g., EDTA), carbohydrates (e.g., dextrin,hydroxyalkylcellulose, and/or hydroxyalkylmethylcellulose), liposomes,stearic acid, liquids (e.g., oils, water, saline, glycerol and/orethanol), wetting or emulsifying agents, pH buffering substances, andthe like.

Formulations for, e.g., parenteral or oral administration, are typicallysolids (for example, a lyophilized powder or cake), liquid solutions,emulsions or suspensions, while inhalable formulations for pulmonaryadministration are generally liquids or powders. Exemplary dosage formsinclude, but are not limited to, tablets, troches, lozenges, aqueous oroil suspensions, non-aqueous solutions, powders, dispersible powders orgranules (including micronized particles or nanoparticles), emulsions,hard or soft capsules, hard or soft liquid-filled capsules, gelatincapsules, syrups, and elixirs. Solid dose formulations, for exampletablets or liquid filled capsules may be uncoated or may be coated byknown techniques including microencapsulation to delay disintegrationand adsorption in the gastrointestinal tract. Solid dose formulationsmay be coated to target delivery to a specific region of the digestivetract. For example, the formulation may be enteric coated to targetdelivery of the formulation to the small intestine, the large intestine,or to the colon. Additional exemplary dosage forms may comprise coatedmicrocapsules or coated microbeads in a suspension or liquid chassis. Insome embodiments, the compound of Formula (I), Formula (II), Formula(III), or Formula (IV) is provided as a dietary (e.g., food or drink)supplement. Dietary supplements are orally dosed and typically comprisevitamins, minerals, herbs or other botanicals, amino acids, enzymes,organ tissues, tissues from glands, or metabolites. In one example, thecompound of Formula (I), Formula (II), Formula (III), or Formula (IV),is provided as a food in the form of a bar.

In some embodiments, the compounds described herein are formulated fororal administration in a lipid-based formulation suitable for lowsolubility compounds. Lipid-based formulations can generally enhance theoral bioavailability of such compounds. As such, the compositioncomprises in some aspects, an amount of a compound described hereintogether with at least one excipient selected from medium chain fattyacids and propylene glycol esters thereof (e.g., propylene glycol estersof edible fatty acids, such as caprylic and capric fatty acids) andphysiologically acceptable surfactants, such as polyoxyl 40 hydrogenatedcastor oil.

In some embodiments, the compound described herein is provided in adelayed release formulation and/or is released in a specific region ofthe digestive tract of an individual. For example, the formulation maybe provided such that the compound is released from an orally dosedformulation in the distal portion of the digestive tract such as theileum or the colon. In certain embodiments, the delayed releaseformulation may release the compounds at a specific pH, or at a range ofpH for the targeted delivery within the digestive tract of anindividual. The compound may be released for example, between pH 6.0 andpH 9.0, between pH 6.5 and pH 8.0, between pH 6.5 and pH 7.5, between pH7.0 and pH 7.5, or between pH 7.0 and pH 8.0.

In certain embodiments a method of the invention optionally comprisesadministering a second agent to the individual. The term “second agent”merely serves to distinguish the agent from the compound of Formula (I),Formula (II), Formula (III), or Formula (IV), and is not meant to limitthe number of additional agents used in a method or denote an order ofadministration. One or more second agents are optionally incorporated inthe composition with the compound of Formula (I), Formula (II), FormulaIII, or Formula (IV), administered concurrently but in separate dosageforms, or administered separately in time.

Exemplary second agents include, but are not limited to, antimicrobials(such as antibiotics that kill bacteria in the gut), agents that improveintestinal motility (such as fiber or psyllium), agents that furtherreduce TMA levels in the gut including sequestering agents (such asactivated charcoal or copper chlorophyllin), and/or agents that furtherreduce the production of TMA metabolites, and agents that improve one ormore aspects of cardiovascular health, such as agents that normalizeblood pressure, decrease vascular inflammation, reduce plateletactivation, normalize lipid abnormalities. In various embodiments, thesecond agent is selected from the group consisting of Omega 3 oil,salicylic acid (aspirin), dimethylbutanol, garlic oil, olive oil, krilloil, Co enzyme Q-10, a probiotic, a prebiotic, a dietary fiber, psylliumhusk, bismuth salts, phytosterols, grape seed oil, green tea extract,vitamin D, an antioxidant (such as vitamin C and vitamin E), turmeric,curcumin, resveratrol, activated charcoal, or copper chlorophyllin.Optionally, the composition comprises dimethylbutanol and/or inhibitorsof the formation of TMA from precursors other than carnitine (e.g.,choline).

Alternatively or in addition, the methods of the disclosure may furthercomprise administration of one or more cardiovascular disease therapies.Examples of therapies include, but are not limited to, statins (e.g.,Lipitor™ (atorvastatin), Pravachol™ (pravastatin), Zocor™ (simvastatin),Mevacor™ (lovastatin), and Lescol™ (fluvastatin)) or other agents thatinterfere with the activity of HMGCoA reductase, nicotinic acid (niacin,which lowers LDL cholesterol levels), fibrates (which lower bloodtriglyceride levels and include, e.g., Bezafibrate (e.g. Bezalip®),Ciprofibrate (e.g. Modalim®), Clofibrate, Gemfibrozil (e.g. Lopid®) andFenofibrate (e.g. TriCor®)), bile acid resins (e.g., Cholestyramine,Colestipol (Colestid), and Cholsevelam (Welchol)), cholesterolabsorption inhibitors (e.g., Ezetimibe (Zetia®, Ezetrol®, Ezemibe®)),phytosterols (e.g., sitosterol (Take Control (Lipton)), sitostanol(Benechol), or stigmastanol), alginates and pectins, lecithin, andnutraceuticals (e.g., extract of green tea and other extracts thatinclude polyphenols, particularly epigallocatechin gallate (EGCG),Cholest-Arrest™ (500 mg garlic and 200 mg lecithin). Cholestaway™ (700mg Calcium carbonate, 170 mg magnesium oxidem 50 μg chromiumpicolinate), Cholest-Off™ (900 mg of plant sterols/stanols), GuggulBolic (750 mg gugulipid (Commiphora mukul gum resin), and Kyolic® (600mg aged garlic extract and 380 mg lecithin)).

In related variations of the preceding embodiments, one or morecompositions comprising a compound of Formula (I), Formula (II), FormulaIII, or Formula (IV), described herein, alone or in combination with oneor more second agents(s), are optionally arranged in a kit or package orunit dose, such as a kit or package or unit dose permittingco-administration of multiple agents. In another aspect, the compositioncomprising a compound of Formula (I), Formula (II), Formula III, orFormula (IV), and the one or more second agents are in admixture. Invarious embodiments, the component(s) of the kit or package or unit doseare packaged with instructions for administering the component(s) to anindividual.

Other aspects and advantages of the present invention will be understoodupon consideration of the following illustrative examples, which are notintended to be limiting in any way.

Example

This example provides an exemplary assay for identifying andcharacterizing compounds that inhibit the formation of TMA fromcarnitine.

Escherichia coli BL21*DE3::pET30a-Ec yeaWX #1 (Ec YeaWX) strain wasgenerated as described below. The contiguous Escherichia coli codingsequence yeaW (equivalent to uniprot ID P0ABR7.1 (YeaW) (SEQ ID NO: 2))and yeaX (equivalent to uniprot ID P76254.1 (YeaX) (SEQ ID NO: 3)) werePCR amplified from Escherichia coli strain K-12 substr. BW25113 genomicDNA. PCR primers (YeaW_Nde_I_fwd2—SEQ ID NO: 4; YeaX_rev2—SEQ ID NO: 5)were designed to create a 5′ NdeI restriction site including the ATGstart codon of yeaW and create a PstI restriction site just 3′ of theyeaX TAG stop codon.

The amplicon was restricted and cloned into the NdeI and PstI sites ofthe plasmid pET30a downstream of the inducible T7 promoter. A blastsearch of the resulting cloned amplicon DNA sequence (SEQ ID NO: 1)corresponded to nucleotide range 1884665 to 1886810 of Escherichia colistr. K-12 substr. MG1655 (NCBI Accession # NC_000913). The construct wastransformed and grown in E. coli BL21 (DE3) and the recombinant yeaWXoverexpressed by addition of isopropyl β-D-1-thiogalactopyranoside(IPTG).

SEQ ID NO Sequence 1 Escherichia coli yeaWX amplicon sequence 2 uniprotID P0ABR7.1, YeaW 3 uniprot ID P76254.1, YeaX 4 YeaW_Nde I_fwd2 5YeaX_rev2A sequence listing that sets forth the nucleotide sequences for SEQ IDNO: 1 to 5 herein is being filed concurrently with the presentapplication as an ASCII text file titled“14120&M_Nucleotide_Sequence_Listing_ST25.” The ASCII text file wascreated on 28 Nov. 2016 and is 10 Kbytes in size. In accordance withMPEP §605.08 and 37 CFR §1.52(e), the subject matter in the ASCII textfile is incorporated herein by reference.

The bacteria were grown aerobically in 50 mL LB broth (Difco #244620; 10g/L Tryptone, 5 g/L yeast extract, 10 g/L NaCl, 50 μg/mL kanamycin), ina 500 mL Erlenmeyer flask. The cultures were inoculated from glycerolstock of BL21*DE3::pET30a-Ec yeaWX #1 strain. Strains were cultured allday at 37° C. with 250 rpm shaking. Two 300 mL Minimal M9 Medium (6 g/LNa₂HPO₄, 3 g/L KH₂PO₄, 0.5 g/L NaCl, 1 g/L NH₄Cl, 0.1 mM CaCl₂, 1 mMMgSO₄, 0.2% Dextrose, 1 mg/L Thiamine, 50 μg/mL kanamycin), in 1 LErlenmeyer flasks, were inoculated with 5 mL of the LB broth day cultureand cultured overnight at 37° C. with 250 rpm shaking. The overnightcultures were used to inoculate twelve 1 L cultures of Minimal M9 mediain 2.8 L fluted Erlenmeyer flasks to an OD 600 nm of 0.05 (typicallyapproximately 28 mLs), which were grown at 37° C. with 250 rpm shakinguntil an OD₆₀₀ of approximately 0.4 was reached. Expression of YeaWX wasinduced with 1 mM IPTG and the induced cultures were further grownovernight at 37° C. with 250 rpm shaking. The biomass was pelleted bycentrifugation at 6000×g for 12 minutes at 4° C. The cell pellet wassuspended in 240 mL of ice-cold 1× Phosphate Buffered Saline (Ca²⁺ andMg²⁺ free). Ninety micrograms of Lysozyme (Sigma# L6876 Lot# SLBG8654V;Sigma-Aldrich Corp., St. Louis, Mo.) was added and incubated with 320rpm shaking for 30 minutes at 4° C. Lysis was achieved via French presswith a 4° C. prechilled 1″ diameter chamber at 1000 psi (high ratio;internal PSI equivalent ˜16000). The lysate was centrifuged at 6,000×gfor 12 minutes at 4° C. to pellet extra debris. Glycerol was added tothe centrifuged lysate supernatant at a final concentration of 15% Aprotein concentration of the centrifuged lysate supernatant wasdetermined by a BCA Protein Assay Kit (Pierce #23225), typically in the2.5 to 4.5 mg/ml range. The centrifuged Ec YeaWX lysate supernatant wasaliquoted into 20 mL volumes and stored frozen at −80° C.

Ec YeaWX lysate was diluted to 2.0 mg/mL protein with 1× Dulbecco'sphosphate buffered saline (DPBS) plus 15% glycerol. Nicotinamide adeninedinucleotide phosphate (NADPH) was added to 250 μM. One hundred andfifty microliters of Ec YeaWX lysate was dispensed into a deep-wellplate (polypropylene, 2 mL volume, Corning Axygen catalogue #P-DW-20-C). Candidate IC₅₀ compounds from TABLE 1 (below) and vehiclecontrol (respective vehicle control of DMSO or water), or controlcompounds (IC₅₀ control, 8-Quinolinol hemisulfate salt (Sigma Catalog#55100)) were added at a1:100 dilution (e.g., 1.5 μL per well). Theplates were agitated on a plate shaker for 1 minute. d9-carnitinechloride (1.5 μL of 5 mM) was added to all wells to reach a finald9-carnitine chloride concentration of 50 μM.

The plates were again agitated on a plate shaker for 1 minute andincubated at 37° C. for two hours. After incubation, 1.5 μL of formicacid was added to each well (final concentration=1% formic acid). Theplates were agitated on a plate shaker for 1 minute and placed on ice.Cell lysate samples were spiked with stable isotope labeled internalstandard (22.5 μL of 6 μg/mL of 13C3-trimethylamine (13C3-TMA) was addedto each sample), then d9-trimethylamine (d9-TMA), trimethylamine (TMA)and 13C3-TMA were isolated from the lysate after protein precipitationas described below. Acetonitrile acidified with 0.1% formic acid, 600μL, was added to each sample which was then centrifuged (2,100 g for 20minutes) to pellet the protein and other precipitates. The supernatantwas removed and analyzed as described below. The TMA, d9-TMA and13C3-TMA in the isolated supernatant samples were subjected to gradientHigh Performance Liquid Chromatography HPLC analysis on a WatersAtlantis HILIC Silica column, from Waters Corp., Milford, Mass., (2.1×50mm, 3 μm particles) with an Atlantis Silica HILIC Sentry guard column,from Waters Corp., Milford, Mass., (100 Å, 3 μm, 2.1 mm×10 mm), 10 mMammonium formate in water with 0.1% formic acid as mobile phase A and0.1% formic acid in acetonitrile as mobile phase B. Detection andquantitation was achieved by tandem mass spectrometry operating undermultiple reaction monitoring (MRM) MS/MS conditions (m/z 60.1→44.1 forTMA, m/z 69.1→49.1 for d9-TMA, m/z 63.0→46.1 for 13C3-TMA). TMA andd9-TMA calibration standards (STD), prepared in 80/20/0.1%acetonitrile/Water/Formic Acid, were used to construct a regressioncurve by plotting the response (peak area TMA/peak area 13C3-TMA) versusconcentration for each standard. The concentrations of TMA and d9-TMA inthe cell lysate were determined by interpolation from the quadratic(1/×2) regression curve.

IC₅₀ measurements of representative compounds of Formula (I) are setforth in TABLE 1.

TABLE 1 TMA Inhibition (IC₅₀, # Compound mol/L) SMILES 12-Methoxy-5-methylphenyl isothiocyanate 6.16E−05 CC1═CC═C(OC)C(N═C═S)═C1

2 tert-Butyl isothiocyanate >0.001 CC(C)(N═C═S)C

3 m-Tolyl isothiocyanate 1.45E−05 CC1═CC(N═C═ S)═CC═C1

4 4-(Methylthio)phenyl isothiocyanate 1.08E−05 CSC1═CC═C(N═C═ S)C═C1

5 Benzyl isothiocyanate 8.37E−06 S═C═NCC1═CC═CC═C1

6 1-pentyl isothiocyanate 3.22E−05 CCCCCN═C═S

7 3-(Diethylamino)propyl isothiocyanate   0.0001458 CCN(CCCN═C═S)CC

8 Cyclohexylmethyl isothiocyanate 3.81E−05 S═C═NCC1CCCCC1

9 2-(4-Chlorophenethyl) isothiocyanate 1.27E−05 ClC1═CC═C(C═C1)CCN═ C═S

10 sec-butyl isothiocyanate   0.0001463 CCC(C)N═C═S

11 Ethyl isothiocyanate 8.22E−05 CCN═C═S

12 Isobutyl isothiocyanate 7.45E−05 CC(CN═C═S)C

13 Butyl isothiocyanate 6.36E−05 CCCCN═C═S

14 Methyl isothiocyanate   0.0001492 CN═C═S

15 Isopropyl isothiocyanate   0.0001996 CC(C)N═C═S

16 1-isothiocyanato-3- methylbutane 7.59E−05 CC(CCN═C═S)C

17 Hexyl isothiocyanate 4.35E−05 CCCCCCN═C═S

18 Phenyl isothiocyanate 6.45E−06 S═C═NC1═CC═CC═C1

19 1-Naphthyl isothiocyanate 1.25E−05 S═C═NC1═C2C═CC═ CC2═CC═C1

20 4-Bromophenyl isothiocyanate 4.72E−06 BrC1═CC═C(C═C1) N═C═S

21 Benzoyl isothiocyanate   0.0001379 O═C(C1═CC═CC═C1)N═ C═S

22 N-Boc-4- isothiocyanatobutylamine 1.77E−05 S═C═NCCCCNC(OC(C) (C)C)═O

23 N-Boc-4- isothiocyanatoaniline 1.57E−05 S═C═NC1═CC═C(NC(OC(C)(C)C)═O)C═C1

24 3-(4-Morpholino)propyl isothiocyanate 6.85E−05 S═C═NCCCN1CCOCC1

25 2-(4-Morpholino)ethyl isothiocyanate 6.90E−05 S═C═NCCN1CCOCC1

26 (S)-(+)-alpha-Methylbenzyl isothiocyanate 7.34E−06 C[C@@H](C1═CC═CC═C1)N═C═S

27 4-Chlorophenyl isothiocyanate 4.57E−06 ClC1═CC═C(N═C═S)C═ C1

28 4-Methoxyphenyl isothiocyanate 5.76E−06 COC1═CC═C(N═C═S)C═ C1

29 exo-2-Norbornyl- isothiocyanate 2.16E−05 S═C═NC1CC2CCC1C2

30 Cyclohexyl isothiocyanate 3.88E−05 S═C═NC1CCCCC1

31 4-Ethylphenyl isothiocyanate 1.73E−05 CCC1═CC═C(N═C═S)C═ C1

32 2-Methoxyphenyl isothiocyanate 3.33E−05 COC1═CC═CC═C1N═C═S

33 2,5-Dimethoxyphenyl isothiocyanate 4.77E−05 COC1═CC═C(OC)C(N═C═ S)═C1

34 3-Methoxyphenyl isothiocyanate 9.59E−06 COC1═CC(N═C═S)═CC═ C1

35 2-Piperidinoethyl isothiocyanate   0.0001043 S═C═NCCN1CCCCC1

36 Ethyl isothiocyanatoacetate 2.999E−05 O═C(OCC)CN═C═S

37 4-Isothiocyanatophenyl 4-pentylbicyclo[2.2.2]octane-1-carboxylate >0.001 O═C(C1(CC2)CCC2(CCCCC) CC1)OC3═CC═C(N═C═S)C═C3

38 Benzhydryl isothiocyanate 3.05E−06 S═C═NC(C1═CC═CC═ C1)C2═CC═CC═C2

39 2,3,4-Tri-O-acetyl-α-D- arabinopyranosyl isothiocyanate 5.012E−05CC(OC1C(N═C═S)OCC (OC(C)═O)C1OC(C)═ O)═O

40 Ethanamine, N,N-diethyl- 2-isothiocyanato 3.199E−04 CCN(CCN═C═S)CC

41 3-isothiocyanato-N,N- dimethyl-1-Propanamine 3.199E−04 CN(C)CCCN═C═S

42 2-Isothiocyanato-N,N- dimethylethanamine 1.637E−04 CN(CCN═C═S)C

43 Pentane, 3-isocyanato- 1.648E−04 CCC(N═C═S)CC

44 Butane, 1-isocyanato-2- methyl- 5.309E−05 CC(CN═C═S)CC

45 2-Isocyanato-N,N,N- trimethylethanaminium iodide 1.154E−04C[N+](CCN═C═ S)(C)C.[I−]

The Example provides exemplary methods of identifying and quantitatingTMA in a sample, as well as screening candidate inhibitory compounds.All compounds in TABLE 1 were found to inhibit the conversion ofcarnitine to TMA.

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “40 mm” is intended to mean“about 40 mm.”Every document cited herein, including any cross referenced or relatedpatent or application and any patent application or patent to which thisapplication claims priority or benefit thereof, is hereby incorporatedherein by reference in its entirety unless expressly excluded orotherwise limited. The citation of any document is not an admission thatit is prior art with respect to any invention disclosed or claimedherein or that it alone, or in any combination with any other referenceor references, teaches, suggests or discloses any such invention.Further, to the extent that any meaning or definition of a term in thisdocument conflicts with any meaning or definition of the same term in adocument incorporated by reference, the meaning or definition assignedto that term in this document shall govern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

What is claimed is:
 1. A method of inhibiting the conversion ofcarnitine to trimethylamine (TMA) by a bacterium comprising: contactingthe bacterium with a compound set forth in Formula (I):

wherein R₁ is selected from cyanate, isocyanate, thiocyanate,isothiocyanate, nitrile, isonitrile, or sulfhydryl; n′ is selected from0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and R₂ is selected from alkyl,branched alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, orsubstituted carbonyl; wherein when R₂ is phenyl, R₂ is substituted with0, 1, or 2 groups independently selected from alkyl, branched alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, halo, or aryl; wherein whenR₂ is heteroalkyl or heterocycloalkyl, the heteroatom(s) are not S;wherein when n′ is 2, R₂ is not unsubstituted phenyl; and including anyacceptable salts or solvates thereof.
 2. The method of claim 1, whereinR₁ is an isothiocyanate.
 3. The method of claim 2, wherein the compoundis sec-butyl isothiocyanate or ethyl isothiocyanate.
 4. The method ofclaim 2, wherein n′ is
 0. 5. The method of claim 4, wherein the compoundis at least one of benzoyl isothiocyanate, 4-bromophenyl isothiocyanate,4-(methylthio)phenyl isothiocyanate, 1-naphthyl isothiocyanate,3-methoxyphenyl isothiocyanate, or 4-methoxyphenyl isothiocyanate. 6.The method of claim 2, wherein n′ is at least
 1. 7. The method of claim6, wherein the compound is at least one of benzyl isothiocyanate,3-diethylaminopropyl isothiocyanate, N-Boc-4-isothiocyanatobutylamine,3-(4-morpholino)propyl isothiocyanate, 2-(4-morpholino)ethylisothiocyanate, or 2-piperidinoethyl isothiocyanate.
 8. The method ofclaim 1 further comprising contacting the bacterium with a second agentthat is at least one of Omega 3 oil, salicylic acid, dimethylbutanol,garlic oil, olive oil, krill oil, Co enzyme Q-10, a probiotic, aprebiotic, dietary fiber, psyllium husk, bismuth salts, phytosterols,grape seed oil, green tea extract, vitamin D, an antioxidant, turmeric,curcumin, resveratrol, activated charcoal, or copper chlorophyllin. 9.The method of claim 1, wherein conversion of carnitine to trimethylamine(TMA) is inhibited by from about 1% to about 100%.
 10. The method ofclaim 1, wherein the bacterium is at least one of Proteus mirabilis,Proteus penneri, Clostridium ljungdahlii, C. scindens, C. aldenense, C.aminobutyricum, Collinsella tanakaei, Anaerococcus vaginalis,Eggerthella lenta, Edwardsiella tarda, Streptococcus dysgalactiae,Desultitobacterium hafniense, Klebsiella variicola, K. pneumonia,Escherichia coli, or E. fergusonii.
 11. The method of claim 1, whereinthe compound has a formula as set forth in Formula (II):

wherein R₂ is as defined for Formula (I); and including any acceptablesalts or solvates thereof.
 12. The method of claim 1, wherein thecompound has a formula as set forth in Formula (III):

wherein n′ and R₂ are as defined for Formula (I); and including anyacceptable salts or solvates thereof.
 13. The method of claim 1, whereinthe compound has a formula as set forth in Formula (IV):

R₃ is hydrogen, alkyl, or aryl; R₄ is aryl; wherein when R₄ is phenyl,R₄ is substituted with 0, 1, or 2 groups independently selected fromalkyl, branched alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, halo,or aryl; and wherein when R₄ is substituted with heteroalkyl orheterocycloalkyl, the heteroatom(s) are not S; and including anyacceptable salts or solvates thereof.
 14. A method of inhibiting theconversion of carnitine to trimethylamine (TMA) in an individualcomprising administering to the individual a compound as set forth inFormula (I):

wherein R₁ is selected from cyanate, isocyanate, thiocyanate,isothiocyanate, nitrile, isonitrile, or sulfhydryl; n′ is selected from0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and R₂ is selected from alkyl,branched alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, orsubstituted carbonyl; wherein when R₂ is phenyl, R₂ is substituted with0, 1, or 2 groups independently selected from alkyl, branched alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, halo, or aryl; wherein whenR₂ is heteroalkyl or heterocycloalkyl, the heteroatom(s) are not S;wherein when n′ is 2, R₂ is not unsubstituted phenyl; and including anyacceptable salts or solvates thereof; wherein the compound isadministered in an amount effective to inhibit formation ofteimethylamine (TMA) from carnitine in the individual.
 15. The method ofclaim 14, wherein R₁ is an isothiocyanate.
 16. The method of claim 15,wherein the compound is sec-butyl isothiocyanate or ethylisothiocyanate.
 17. The method of claim 14, wherein n′ is
 0. 18. Themethod of claim 17, wherein the compound is at least one of benzoylisothiocyanate, 4-bromophenyl isothiocyanate, 4-(methylthio)phenylisothiocyanate, 1-naphthyl isothiocyanate, 3-methoxyphenylisothiocyanate, or 4-methoxyphenyl isothiocyanate.
 19. The method ofclaim 14, wherein n′ is at least
 1. 20. The method of claim 19, whereinthe compound is at least one of benzyl isothiocyanate,3-diethylaminopropyl isothiocyanate, N-Boc-4-isothiocyanatobutylamine,3-(4-morpholino)propyl isothiocyanate, 2-(4-morpholino)ethylisothiocyanate, or 2-piperidinoethyl isothiocyanate.
 21. The method ofclaim 14 further comprising administering to the individual a secondagent that is at least one of Omega 3 oil, salicylic acid,dimethylbutanol, garlic oil, olive oil, krill oil, Co enzyme Q-10, aprobiotic, a prebiotic, dietary fiber, psyllium husk, bismuth salts,phytosterols, grape seed oil, green tea extract, vitamin D, anantioxidant, turmeric, curcumin, resveratrol, activated charcoal, orcopper chlorophyllin.
 22. A method of improving a condition associatedwith the conversion of carnitine to trimethylamine (TMA) in anindividual comprising administering to the individual a compound setforth in Formula (I):

wherein R₁ is selected from cyanate, isocyanate, thiocyanate,isothiocyanate, nitrile, isonitrile, or sulfhydryl; n′ is selected from0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and R₂ is selected from alkyl,branched alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, orsubstituted carbonyl; wherein when R₂ is phenyl, R₂ is substituted with0, 1, or 2 groups independently selected from alkyl, branched alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, halo, or aryl; wherein whenR₂ is heteroalkyl or heterocycloalkyl, the heteroatom(s) are not S;wherein when n′ is 2, R₂ is not unsubstituted phenyl; and including anyacceptable salts or solvates thereof; wherein the compound isadministered in an amount effective to treat or prevent the disease orcondition associated with carnitine-related trimethylamine (TMA) in theindividual.
 23. The method of claim 22, wherein the condition associatedwith carnitine-related trimethylamine is at least one of acardiovascular disease, reduced or impaired kidney function, chronickidney disease, or diabetes mellitus.